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Product Description:Mesenchymal Stem Cel Osteogenic Differentiation Medium consists of optimized Mesenchymal Stem Cell Osteogenic
Differentiation Basal Medium, pre-selected Fetal Bovine Serum and supplements. This product has been developed for the
optimal differentiation of Mesenchymal Stem Cells into osteoblasts. The product is intended for laboratory research use only,
not for drug, house hold, or other uses.
Kit Components: Mesenchymal Stem Cel Osteogenic Differentiation Basal Medium 175 mL
Mesenchymal Stem Cel -Qualified Fetal Bovine Serum 20 mL
Instructions for Use: Preparation of complete medium
Prior to use, thaw Mesenchymal Stem Cel -Qualified Fetal Bovine Serum under refrigeration (2 to 8°C) over night or until
completely thawed. Gently swirl the bottle to ensure homogeneity. Mesenchymal Stem Cell-Qualified Fetal Bovine Serum
has been heatinactivated and is ready to use after thawing.
Note: The thawed serum may contain some flocculent precipitates. The presence of these substances in serum does not alter the performance characteristics of the product. It is not recommended to filter the serum to remove these precipitates. Doing so may result in the loss of some serum nutrients.
About 30 minutes prior to use, thaw Ascorbate, β-Glycerophosphate, Penicillin-Streptomycin solution and Glutamine
solution at room temperature. Gently invert the vials several times to ensure homogeneity.
Note: Centrifuge the vials briefly at low speed (5,000g) before removing the caps to ensure recovery of entire content.
About 10 minutes prior to use, thaw Dexamethasone at room temperature.
Note: Centrifuge the vial briefly at low speed (5,000g) before removing the cap to ensure recovery of entire content.
Disinfect with 70% v/v ethanol the external surfaces of the bottles/vials for every component in the kit. Allow ethanol to
In a laminar flow hood aseptically open the bottles/vials.
Transfer the entire amount of Ascorbate, β-Glycerophosphate, Mesenchymal Stem Cell-Qualified Fetal Bovine Serum,
Penicillin-Streptomycin solution and Glutamine solution into Mesenchymal Stem Cell Osteogenic Differentiation Basal
Rinse each vial/bottle with the medium and transfer the rinse medium back to the bottle of basal medium.
To transfer the entire amount of Dexamethasone, add 0.5 mL medium to the vial, mix by pipeting and then transfer the
mixture back to the bottle of basal medium as much as possible.
Gently swirl the fully supplemented complete medium to ensure a homogeneous mixture. The complete medium is now
Note: Although each component in this kit is supplied sterile, it is strongly recommended to filter the fully supplemented complete medium. Gelatin Coating of Tissue Culture Vessels:
Add sufficient 0.1% Gelatin Solution into the culture vessel to completely cover its base.
Swirl until Gelatin Solution coats entire base of vessel. Let sit for at least 30 minutes at room temperature.
Aspirate off all of the Gelatin Solution and allow the remainder to evaporate by leaving the vessel sitting open in the hood
Osteogenesis protocol (for 6-well tissue culture plate):
Mesenchymal Stem Cells are cultured in Mesenchymal Stem Cell Growth Medium (growth medium thereafter) at 37°C in a
When cells are approximately 80-90% confluent, they can be dissociated with Trypsin-EDTA.
Mesenchymal Stem Cel s are replated in growth medium at 3×103 cells/cm2 in 6-well tissue culture plates pre-coated with
Incubate the cells at 37°C in a 5% CO2 humidified incubator.
After 24 hours, carefully aspirate off the growth medium from each well and add 2 mL Mesenchymal Stem Cell Osteogenic
Refeed cells every 3 days for 2-3 weeks by completely replace the medium with fresh Mesenchymal Stem Cell Osteogenic
After 2-3 week differentiation, cells can be fixed and stained with Alizarin red.
Alizarin Red Stain Analysis:
After differentiating, remove Osteogenic Differentiation Medium from well and rinse with 1×PBS. Fix cells with 2ml of 4%
Then rinse wells twice with 1×PBS and stain cells with 1ml alizarin red working solution for 3 to 5 minutes.
Rinse wells 2-3 times with 1×PBS, then visualize under light microscope and capture images.
Stability/Storage: All products should be stored in the dark. Mesenchymal Stem Cell Osteogenic Differentiation Basal Medium is stable at 2 to 8°C
for up to one year. Other components are stable at -20°C for up to two years. These products should be discarded beyond the
labeled expiration date. Once prepared, the fully supplemented complete medium can be stored for up to one month when
stored in the dark at 2 to 8°C. For optimal performance, repeated warm-cooling and freeze-thawing should be avoided.
Standard evaluation includes: 1. Sterility test (bacteria, fungi, mold and mycoplasma)

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